Institut für Chemie, Technische Universität Berlin, Müller-Breslau-Straße 10, 10623 Berlin, Germany.
Institute of Biological Chemistry, Academia Sinica, Taipei 116, Taiwan.
Molecules. 2020 Sep 25;25(19):4418. doi: 10.3390/molecules25194418.
In protein engineering and synthetic biology, pyrrolysyl-tRNA synthetase (PylRS), with its cognate tRNA, is one of the most popular tools for site-specific incorporation of non-canonical amino acids (ncAAs). Numerous orthogonal pairs based on engineered PylRS variants have been developed during the last decade, enabling a substantial genetic code expansion, mainly with aliphatic pyrrolysine analogs. However, comparatively less progress has been made to expand the substrate range of PylRS towards aromatic amino acid residues. Therefore, we set to further expand the substrate scope of orthogonal translation by a semi-rational approach; redesigning the PylRS efficiency. Based on the randomization of residues from the binding pocket and tRNA binding domain, we identify three positions (V401, W417 and S193) crucial for ncAA specificity and enzyme activity. Their systematic mutagenesis enabled us to generate PylRS variants dedicated to tryptophan (such as β-(1-Azulenyl)-l-alanine or 1-methyl-l-tryptophan) and tyrosine (mainly halogenated) analogs. Moreover, our strategy also significantly improves the orthogonal translation efficiency with the previously activated analog 3-benzothienyl-l-alanine. Our study revealed the engineering of both first shell and distant residues to modify substrate specificity as an important strategy to further expand our ability to discover and recruit new ncAAs for orthogonal translation.
在蛋白质工程和合成生物学中，吡咯赖氨酰-tRNA 合成酶（PylRS）及其对应的 tRNA 是用于特异性掺入非天然氨基酸（ncAA）的最流行的工具之一。在过去的十年中，已经开发出了许多基于工程化 PylRS 变体的正交对，从而实现了遗传密码的实质性扩展，主要是利用脂肪族吡咯赖氨酸类似物。然而，在将 PylRS 的底物范围扩展到芳香族氨基酸残基方面，进展相对较少。因此，我们通过半理性方法进一步扩展正交翻译的底物范围；重新设计 PylRS 的效率。基于结合口袋和 tRNA 结合结构域中残基的随机化，我们确定了三个对 ncAA 特异性和酶活性至关重要的位置（V401、W417 和 S193）。对这些位置的系统诱变使我们能够生成专门用于色氨酸（如β-（1-薁基）-l-丙氨酸或 1-甲基-l-色氨酸）和酪氨酸（主要是卤代）类似物的 PylRS 变体。此外，我们的策略还显著提高了先前激活的类似物 3-苯并噻吩基-l-丙氨酸的正交翻译效率。我们的研究揭示了修饰底物特异性的第一壳层和远程残基的工程化是进一步扩展我们发现和招募新的正交翻译 ncAA 的能力的重要策略。
